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- Hendriks, GJ, et al.
(författare)
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NASC-seq monitors RNA synthesis in single cells
- 2019
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10:1, s. 3138-
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Tidskriftsartikel (refereegranskat)abstract
- Sequencing of newly synthesised RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesised and pre-existing RNA simultaneously in single cells. We validate the method on pre-labelled RNA, and by demonstrating that more newly synthesised RNA was detected for genes with known high mRNA turnover. Monitoring RNA synthesis during Jurkat T-cell activation with NASC-seq reveals both rapidly up- and down-regulated genes, and that induced genes are almost exclusively detected as newly transcribed. Moreover, the newly synthesised and pre-existing transcriptomes after T-cell activation are distinct, confirming that NASC-seq simultaneously measures gene expression corresponding to two time points in single cells. Altogether, NASC-seq enables precise temporal monitoring of RNA synthesis at single-cell resolution during homoeostasis, perturbation responses and cellular differentiation.
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- Johnsson, P, et al.
(författare)
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Transcriptional kinetics and molecular functions of long noncoding RNAs
- 2022
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Ingår i: Nature genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 54:43, s. 306-
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Tidskriftsartikel (refereegranskat)abstract
- An increasing number of long noncoding RNAs (lncRNAs) have experimentally confirmed functions, yet little is known about their transcriptional dynamics and it is challenging to determine their regulatory effects. Here, we used allele-sensitive single-cell RNA sequencing to demonstrate that, compared to messenger RNAs, lncRNAs have twice as long duration between two transcriptional bursts. Additionally, we observed increased cell-to-cell variability in lncRNA expression due to lower frequency bursting producing larger numbers of RNA molecules. Exploiting heterogeneity in asynchronously growing cells, we identified and experimentally validated lncRNAs with cell state-specific functions involved in cell cycle progression and apoptosis. Finally, we identified cis-functioning lncRNAs and showed that knockdown of these lncRNAs modulated the nearby protein-coding gene’s transcriptional burst frequency or size. In summary, we identified distinct transcriptional regulation of lncRNAs and demonstrated a role for lncRNAs in the regulation of mRNA transcriptional bursting.
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- Larsson, AJM, et al.
(författare)
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Transcriptional bursts explain autosomal random monoallelic expression and affect allelic imbalance
- 2021
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Ingår i: PLoS computational biology. - : Public Library of Science (PLoS). - 1553-7358. ; 17:3, s. e1008772-
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Tidskriftsartikel (refereegranskat)abstract
- Transcriptional bursts render substantial biological noise in cellular transcriptomes. Here, we investigated the theoretical extent of allelic expression resulting from transcriptional bursting and how it compared to the amount biallelic, monoallelic and allele-biased expression observed in single-cell RNA-sequencing (scRNA-seq) data. We found that transcriptional bursting can explain the allelic expression patterns observed in single cells, including the frequent observations of autosomal monoallelic gene expression. Importantly, we identified that the burst frequency largely determined the fraction of cells with monoallelic expression, whereas the burst size had little effect on monoallelic observations. The high consistency between the bursting model predictions and scRNA-seq observations made it possible to assess the heterogeneity of a group of cells as their deviation in allelic observations from the expected. Finally, both burst frequency and size contributed to allelic imbalance observations and reinforced that studies of allelic imbalance can be confounded from the inherent noise in transcriptional bursting. Altogether, we demonstrate that allele-level transcriptional bursting renders widespread, although predictable, amounts of monoallelic and biallelic expression in single cells and cell populations.
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- Ziegenhain, C, et al.
(författare)
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Molecular spikes: a gold standard for single-cell RNA counting
- 2022
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Ingår i: Nature methods. - : Springer Science and Business Media LLC. - 1548-7105 .- 1548-7091. ; 19:5, s. 560-
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Tidskriftsartikel (refereegranskat)abstract
- Single-cell sequencing methods rely on molecule-counting strategies to account for amplification biases, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes—RNA spike-ins containing built-in unique molecular identifiers (UMIs) that we use to identify critical experimental and computational conditions for accurate RNA counting in single-cell RNA-sequencing (scRNA-seq). Using molecular spikes, we uncovered impaired RNA counting in methods that were not informative for cellular RNA abundances due to inflated UMI counts. We further leverage molecular spikes to improve estimates of total endogenous RNA amounts in cells, and introduce a strategy to correct experiments with impaired RNA counting. The molecular spikes and the accompanying R package UMIcountR (https://github.com/cziegenhain/UMIcountR) will improve the validation of new methods, better estimate and adjust for cellular mRNA amounts and enable more indepth characterization of RNA counting in scRNA-seq.
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